Yang Li

Introduction: Cardiovascular–kidney–metabolic (CKM) syndrome represents an emerging systemic disorder characterized by intertwined metabolic dysfunction, chronic kidney disease, and cardiovascular injury, yet robust preclinical models and mechanistic insights remain limited. Methods: Here, we established a progressive rat model of CKM syndrome by combining high-fat/high-sucrose exposure with adenine-induced renal stress, capturing temporal changes from early metabolic dysregulation to advanced multi-organ injury. Integrative multi-omics analyses incorporating network toxicology, single-cell RNA sequencing, and spatial transcriptomics were performed to investigate molecular features associated with CKM progression. Finally, AI-assisted virtual screening coupled with molecular docking was conducted to identify potential multi-target therapeutic candidates. Results: Longitudinal phenotyping revealed a temporal pattern in which early metabolic abnormalities preceded more prominent renal and cardiovascular impairment. Integrative multi-omics analyses incorporating network toxicology, single-cell RNA sequencing, and spatial transcriptomics consistently converged on lipid metabolic dysregulation as a prominent molecular feature associated with CKM progression. PPARγ, ESR1, and FASN were identified as candidate regulatory nodes associated with CKM-related lipid remodeling. Single-cell analyses revealed enrichment of lipid metabolic programs in renal epithelial compartments, particularly proximal tubular cells, suggesting their potential involvement in CKM-associated renal metabolic remodeling. Spatial transcriptomics further revealed patterns consistent with ectopic adipocyte infiltration, lipid-associated niche remodeling, increased PPARγ/FASN expression, and reduced ESR1 expression in diseased kidneys. AI-assisted virtual screening coupled with molecular docking identified BRD-K26818574 as a potential multi-target therapeutic candidate. Discussion: Collectively, this study establishes a translational CKM model and highlights renal lipotoxic remodeling as a potential therapeutic target in CKM progression.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related mortality worldwide, with limited therapeutic options and poor clinical outcomes. Mounting evidence suggests that targeting cancer-specific metabolic and redox adaptations represents a promising therapeutic strategy. Peroxiredoxin 1 (PRDX1), a key antioxidant enzyme that is frequently overexpressed in HCC, enables tumor cells to neutralize excessive reactive oxygen species (ROS), thereby sustaining survival and conferring therapeutic resistance. In this study, using human hepatocellular carcinoma HepG2 cells as an in vitro model, we identify ginsenoside Rg5 (Rg5) as a previously unrecognized small-molecule inhibitor of PRDX1. Structural and functional analyses demonstrate that Rg5 directly binds to the Asn145 residue of PRDX1, effectively suppressing its peroxidase activity. Mechanistically, this inhibition disrupts ROS detoxification in HepG2 cells, leading to mitochondrial ROS accumulation, activation of the intrinsic apoptotic pathway, and consequent HepG2 cell death. Additionally, Rg5 not only suppresses HepG2 cell survival but also acts synergistically with doxorubicin, a first-line chemotherapeutic agent, to markedly enhance antitumor efficacy and potentially mitigate chemoresistance. Collectively, these findings suggest that PRDX1 inhibition may represent a broadly exploitable vulnerability in liver cancer and establish Rg5 as a promising candidate for developing targeted and combinatorial therapies against HCC.

Ginsenoside Rh2 (G-Rh2), a rare ginsenoside isolated from red ginseng, has considerable anti-cancer activity and induces apoptosis in a variety of cancer cells, but its activity in esophageal cancer cells is unclear. In this study, we examined the cytotoxic activity of (20S) G-Rh2 in highly differentiated esophageal squamous ECA109 cells and poorly differentiated esophageal squamous TE-13 cells. (20S) G-Rh2 exerted intense cytotoxicity in ECA109 and TE-13 cells with an IC50 of 2.9 and 3.7 μg/mL, respectively. After treatment with G-Rh2, Bcl-2, and Bcl-xL, the two main anti-apoptosis Bcl-2 family proteins upregulated, and Bax and Bak, the two key pro-apoptosis proteins translocated to mitochondria in both cell lines. At the same time, cytochrome c and Smac released from mitochondria, followed by caspase-9 activation, indicating that a mitochondria-mediated intrinsic apoptosis pathway was activated in both cell lines upon treatment with (20S) G-Rh2. It is noteworthy that (20S) G-Rh2 upregulated the transcription and protein expression of two death receptors, Fas and DR5, and subsequently activated Caspase-8 in the TE-13 cells but not in the ECA109 cells. Taken together, we demonstrated the potent anti-esophageal cancer cell activity of (20S) G-Rh2 and showed its working mechanism in two differentiated esophageal cancer cells, which can provide important evidence for developing an effective strategy for anti-esophageal cancer treatment.

(20S) ginsenoside Rh2 (G-Rh2), a major bioactive metabolite of ginseng, effectively inhibits the survival and proliferation of human liver cancer cells. However, its molecular targets and working mechanism remain largely unknown. Excitingly, we screened out heat shock protein 90 alpha (HSP90A), a key regulatory protein associated with liver cancer, as a potential target of (20S) G-Rh2 by phage display analysis and mass spectrometry. The molecular docking and thermal shift analyses demonstrated that (20S) G-Rh2 directly bound to HSP90A, and this binding was confirmed to inhibit the interaction between HSP90A and its co-chaperone, cell division cycle control protein 37 (Cdc37). It is well-known that the HSP90A-Cdc37 system aids in the folding and maturation of cyclin-dependent kinases (CDKs). As expected, CDK4 and CDK6, the two G₀-G₁ phase promoting kinases as well as CDK2, a key G₁-S phase transition promoting kinase, were significantly downregulated with (20S) G-Rh2 treatment, and these downregulations were mediated by the proteasome pathway. In the same condition, the cell cycle was arrested at the G₀-G₁ phase and cell growth was inhibited significantly by (20S) G-Rh2 treatment. Taken together, this study for the first time reveals that (20S) G-Rh2 exerts its anti-tumor effect by targeting HSP90A and consequently disturbing the HSP90A-Cdc37 chaperone system. HSP90A is frequently overexpressed in human hepatoma cells and the higher expression is closely correlated to the poor prognosis of liver cancer patients. Thus, (20S) G-Rh2 might become a promising alternative drug for liver cancer therapy.

Signal transducers and activators of transcription 3 (STAT3) acts as a transcriptional signal transducer, converting cytokine stimulation into specific gene expression. In tumor cells, aberrant activation of the tyrosine kinase pathway leads to excessive and continuous activation of STAT3, which provides further signals for tumor cell growth and surrounding angiogenesis. In this process, the tumor-associated protein Annexin A2 interacts with STAT3 and promotes Tyr705 phosphorylation and STAT3 transcriptional activation. In this study, we found that (20S) ginsenoside Rh2 (G-Rh2), a natural compound inhibitor of Annexin A2, inhibited STAT3 activity in HepG2 cells. (20S) G-Rh2 interfered with the interaction between Annexin A2 and STAT3, and inhibited Tyr705 phosphorylation and subsequent transcriptional activity. The inhibitory activity of STAT3 leaded to the negative regulation of the four VEGFs, which significantly reduced the enhanced growth and migration ability of HUVECs in co-culture system. In addition, (20S)G-Rh2 failed to inhibit STAT3 activity in cells overexpressing (20S)G-Rh2 binding-deficient Annexin A2-K301A mutant, further proving Annexin A2-mediated inhibition of STAT3 by (20S)G-Rh2. These results indicate that (20S)G-Rh2 is a potent inhibitor of STAT3, predicting the potential activity of (20S)G-Rh2 in targeted therapy applications.

Ginsenoside Rk1 and Rg5 are minor ginseng saponins that have received more attention recently because of their high oral bioavailability. Each of them can effectively inhibit the survival and proliferation of human liver cancer cells, but the underlying mechanism remains largely unknown. Network pharmacology and bioinformatics analysis demonstrated that G-Rk1 and G-Rg5 yielded 142 potential targets, and shared 44 putative targets associated with hepatocellular carcinoma. Enrichment analysis of the overlapped genes showed that G-Rk1 and G-Rg5 may induce apoptosis of liver cancer cells through inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signal pathways. Methyl thiazolyl tetrazolium (MTT) assay was used to confirm the inhibition of cell viability with G-Rk1 or G-Rg5 in highly metastatic human cancer MHCC-97H cells. We evaluated the apoptosis of MHCC-97H cells by using flow cytometry and 4′,6-diamidino-2-phenylindole (DAPI) staining. The translocation of Bax/Bak led to the depolarization of mitochondrial membrane potential and release of cytochrome c and Smac. A sequential activation of caspase-9 and caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed after that. The levels of anti-apoptotic proteins were decreased after treatment of G-Rk1 or G-Rg5 in MHCC-97H cells. Taken together, G-Rk1 and G-Rg5 promoted the endogenous apoptotic pathway in MHCC-97H cells by targeting and regulating some critical liver cancer related genes that are involved in the signal pathways associated with cell survival and proliferation.

Human serum albumin (HSA) is an important component of plasma, which has the functions of maintaining colloid osmotic pressure and capillary membrane stability, promoting blood circulation, and anti-oxidation. Three-dimensional structure of HSA determines its ability to bind and transport hormones and other substances. In this study we examined the interactions between HSA and ginsenoside Rg3, Rg5, Rk1, Rh2, and Rh4, which are the main cytotoxic ginsenosides extracted from red ginseng. Heat transfer generated by the specific interaction between HSA and each ginsenoside was measured using isothermal titration calorimetry (ITC) assay, which demonstrated that all these 5 ginsenosides bound to HSA with binding constants of 3.25, 1.89, 6.04, 2.07, and 5.17 × 10⁵ M⁻¹, respectively. Molecular docking also displayed that these ginsenosides interact with HSA at different sites of the HSA surface. Importantly, cell viability assay showed that the cytotoxicity of these ginsenosides reduced significantly at the presence of HSA in human vascular endothelial cells (HUVEC). Taken together, this study reveals the mechanism by which these ginsenosides are transported in vivo by not causing damage in vascular endothelium, and also suggests HSA might be an ideal carrier help to transport and execute these ginsenoside functions in human body.

(1) Background: Epithelial-mesenchymal transition (EMT) is an essential step for cancer metastasis; targeting EMT is an important path for cancer treatment and drug development. NF-κB, an important transcription factor, has been shown to be responsible for cancer metastasis by enhancing the EMT process. Our previous studies showed that (20S)Ginsenoside Rh2 (G-Rh2) inhibits NF-κB activity by targeting Anxa2, but it is still not known whether this targeted inhibition of NF-κB can inhibit the EMT process. (2) Methods: In vivo (20S)G-Rh2-Anxa2 interaction was assessed by cellular thermal shift assay. Protein interaction was determined by immuno-precipitation analysis. NF-κB activity was determined by dual luciferase reporter assay. Gene expression was determined by RT-PCR and immuno-blot. EMT was evaluated by wound healing and Transwell assay and EMT regulating gene expression. (3) Results: Anxa2 interacted with the NF-κB p50 subunit, promoted NF-κB activation, then accelerated mesenchymal-like gene expression and enhanced cell motility; all these cellular processes were inhibited by (20S)G-Rh2. In contrast, these (20S)G-Rh2 effect were completely eliminated by overexpression of Anxa2-K301A, an (20S)G-Rh2-binding-deficient mutant of Anxa2. (4) Conclusion: (20S)G-Rh2 inhibited NF-κB activation and related EMT by targeting Anxa2 in MDA-MB-231 cells.